Goals

Load in phyloseq data with rooted tree.
Evaluate sequencing depth and remove sample.
Normalize the read counts between samples.
Calculate community dissimilarities. Numbers between 0 and 1. If 0, completely similar versus if they are 1, then they’re completely dissimilar.
1. Sorensen: Shared Species as a binary value: Abundance-unweighted
2. Bray-Curtis: Shared Abundant species: Abundance-weighted
3. (Abundance-)Weighted UNIFRAC: Consider Abundant Species and where they fall on the tree
Visualize the community data with two unconstrained Ordinations:
1. PCoA: Linear Method. Eigenvalue = how much variation is explained by each axis. Choose to view axis 1, 2, 3, etc. and plot them together.
2. NMDS: Non-linear. Smush multiple Dimensions into 2 or 3 axes. Need to report Stress value (ideally <0.15).
Run statistics with PERMANOVA and betadispR.

Setup

Set the seed

# Any number can be chosen 
set.seed(238428)

Load Libraries

#install.packages("vegan")
pacman::p_load(tidyverse, devtools, phyloseq, patchwork, vegan,
               install = FALSE)

# Load colors 
n_colors <- c(
  "0" = "#D9CC3C",
  "150" = "#A0E0BA",
  "300" = "coral2")
  
growth_stage_colors <- c (
  "30" = "lightblue",
  "75" = "darkblue")

planted_colors <- c (
  "Unplanted" = "springgreen",
  "Planted" = "purple")

Load Data

# Load in rooted phylogenetic tree! 
load("data/03_Phylogenetic_Tree/phytree_preprocessed_physeq.RData")
midroot_physeq_rm18392

## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 31491 taxa and 35 samples ]
## sample_data() Sample Data:       [ 35 samples by 12 sample variables ]
## tax_table()   Taxonomy Table:    [ 31491 taxa by 9 taxonomic ranks ]
## phy_tree()    Phylogenetic Tree: [ 31491 tips and 31490 internal nodes ]

unrooted_physeq_rm18392

## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 31491 taxa and 35 samples ]
## sample_data() Sample Data:       [ 35 samples by 12 sample variables ]
## tax_table()   Taxonomy Table:    [ 31491 taxa by 9 taxonomic ranks ]
## phy_tree()    Phylogenetic Tree: [ 31491 tips and 31489 internal nodes ]

Explore Read Counts

Raw read depth

# Calculate the total number of reads per sample. 
raw_TotalSeqs_df <- 
  midroot_physeq_rm18392 %>%
  # calculate the sample read sums 
  sample_sums() %>%
  data.frame()
# name the column 
colnames(raw_TotalSeqs_df)[1] <- "TotalSeqs"
  
head(raw_TotalSeqs_df)

##             TotalSeqs
## SRR21916202     29262
## SRR21916203     38724
## SRR21916205     42736
## SRR21916206     37619
## SRR21916207     35134
## SRR21916208     30827

# make histogram of raw reads 
raw_TotalSeqs_df %>%
  ggplot(aes(x = TotalSeqs)) + 
  geom_histogram(bins = 50 ) + 
  scale_x_continuous(limits = c(0, 30000)) + 
  labs(title = "Raw Sequencing Depth Distribution") + 
  theme_classic()

## Warning: Removed 23 rows containing non-finite outside the scale range
## (`stat_bin()`).

## Warning: Removed 2 rows containing missing values or values outside the scale range
## (`geom_bar()`).

Remove lowly seq sample

raw_rooted_physeq <- 
  midroot_physeq_rm18392 %>%
  # remove lowly seq sample that was outlier in alpha diversity analysis
  subset_samples(names != "SRR21916204") %>%
  # any asvs unique to this sample will also be removed 
  prune_taxa(taxa_sums(.) > 0, .)

# Inspect 
raw_rooted_physeq

## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 31491 taxa and 35 samples ]
## sample_data() Sample Data:       [ 35 samples by 12 sample variables ]
## tax_table()   Taxonomy Table:    [ 31491 taxa by 9 taxonomic ranks ]
## phy_tree()    Phylogenetic Tree: [ 31491 tips and 31490 internal nodes ]

# what is the minimum number of sequences 
raw_rooted_physeq %>%
  sample_sums() %>%
  min()

## [1] 17544

Normalize read counts

### scale_reads function
#################################################################################### 
# Function to scale reads: http://deneflab.github.io/MicrobeMiseq/ 
# Scales reads by 
# 1) taking proportions
# 2) multiplying by a given library size of n
# 3) rounding 
# Default for n is the minimum sample size in your library
# Default for round is floor

matround <- function(x){trunc(x+0.5)}

scale_reads <- function(physeq, n = min(sample_sums(physeq)), round = "round") {
  
  # transform counts to n
  physeq.scale <- transform_sample_counts(physeq, function(x) {(n * x/sum(x))})
  
  # Pick the rounding functions
  if (round == "floor"){
    otu_table(physeq.scale) <- floor(otu_table(physeq.scale))
  } else if (round == "round"){
    otu_table(physeq.scale) <- round(otu_table(physeq.scale))
  } else if (round == "matround"){
    otu_table(physeq.scale) <- matround(otu_table(physeq.scale))
  }
  
  # Prune taxa and return new phyloseq object
  physeq.scale <- prune_taxa(taxa_sums(physeq.scale) > 0, physeq.scale)
  return(physeq.scale)
}

Scale the reads and check the distribution of the seq depth

This is where one might decide use rarefaction to normalize data.

min(sample_sums(raw_rooted_physeq))

## [1] 17544

# Scale reads by the above function
scaled_rooted_physeq <- 
  raw_rooted_physeq %>%
  scale_reads(round = "matround")

# Calculate the read depth 
scaled_TotalSeqs_df <- 
  scaled_rooted_physeq %>%
  sample_sums() %>%
  data.frame()
colnames(scaled_TotalSeqs_df)[1] <-"TotalSeqs"

# Inspect
head(scaled_TotalSeqs_df)

##             TotalSeqs
## SRR21916202     17546
## SRR21916203     17459
## SRR21916205     17522
## SRR21916206     17387
## SRR21916207     17027
## SRR21916208     17618

# Check the range of the data 
min_seqs <- min(scaled_TotalSeqs_df$TotalSeqs); min_seqs

## [1] 17027

max_seqs <- max(scaled_TotalSeqs_df$TotalSeqs); max_seqs

## [1] 17869

# range
max_seqs - min_seqs

## [1] 842

# Plot Histogram 
scaled_TotalSeqs_df %>%
  ggplot(aes(x = TotalSeqs)) + 
  geom_histogram(bins = 50) + 
  scale_x_continuous(limits = c(0, 30000)) + 
  labs(title = "Scaled Sequencing Depth at 2194") + 
  theme_classic()

## Warning: Removed 2 rows containing missing values or values outside the scale range
## (`geom_bar()`).

Cacluate & Visualize community dissimilarity

Exploratory analyses from Paliy & Shankar (2016) paper, which is using unconstrained ordination methods like PCoA.

PCoA

Sorensen

# Calculate sorensen dissimilarity: Abundance-unweighted of shared taxa
scaled_soren_pcoa <-  
  ordinate(
    physeq = scaled_rooted_physeq,
    method = "PCoA",
    distance = "bray", binary = TRUE)

#str(scaled_soren_pcoa)

# Plot the ordination
n_colors <- as.factor(n_colors)
soren_n_pcoa <- plot_ordination(
  physeq = scaled_rooted_physeq,
  ordination = scaled_soren_pcoa,
  color = as.factor("n_level"),
  title = "Sorensen PCoA") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(n_level))) +
  scale_color_manual(values = as.factor(n_colors)) + 
  theme_bw()
# Show the plot 
soren_n_pcoa

# Plot for comparison between planted vs. unplanted status
# Plot the ordination
planted_colors <- as.factor(planted_colors)
soren_planted_pcoa <- plot_ordination(
  physeq = scaled_rooted_physeq,
  ordination = scaled_soren_pcoa,
  color = as.factor("planted"),
  title = "Sorensen PCoA") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(planted))) +
  scale_color_manual(values = as.factor(planted_colors)) + 
  theme_bw()
# Show the plot 
soren_planted_pcoa

Much clearer presence/absence separation based on planted vs. unplanted status.

Bray

# Calculate the BC distance
scaled_BC_pcoa <- 
  ordinate(
    physeq = scaled_rooted_physeq,
    method = "PCoA",
    distance = "bray")

# Plot the PCoA
bray_n_pcoa <- plot_ordination(
  physeq = scaled_rooted_physeq,
  ordination = scaled_BC_pcoa,
  color = as.factor("n_level"),
  title = "Bray-Curtis PCoA") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(n_level))) +
  scale_color_manual(values = as.factor(n_colors)) + 
  theme_bw()
# Show the plot 
bray_n_pcoa

# Plot for comparison between planted vs. unplanted status
# Plot the PCoA
bray_planted_pcoa <- plot_ordination(
  physeq = scaled_rooted_physeq,
  ordination = scaled_BC_pcoa,
  color = as.factor("planted"),
  title = "Bray-Curtis PCoA") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(planted))) +
  scale_color_manual(values = as.factor(planted_colors)) + 
  theme_bw()
# Show the plot 
bray_planted_pcoa

More variance can be explained by the axis of the abundance-weighted ordination: 20% for Bray-Curtis vs. only 10.3% for Sorensen ordination. Clear separations based on n-level are not evident, but separations based on plant presence are!

Weighted Unifrac

# Calculate the BC distance
scaled_wUNI_pcoa <- 
  ordinate(
    physeq = scaled_rooted_physeq,
    method = "PCoA",
    distance = "wunifrac")

# Plot the PCoA
wUNI_n_pcoa <- 
  plot_ordination(
    physeq = scaled_rooted_physeq,
    ordination = scaled_wUNI_pcoa,
    color = as.factor("n_level"),
    title = "Weighted Unifrac PCoA") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(n_level))) +
  scale_color_manual(values = as.factor(n_colors)) + 
  theme_bw()
wUNI_n_pcoa

wUNI_planted_pcoa <- 
  plot_ordination(
    physeq = scaled_rooted_physeq,
    ordination = scaled_wUNI_pcoa,
    color = as.factor("planted"),
    title = "Weighted Unifrac PCoA") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(planted))) +
  scale_color_manual(values = as.factor(planted_colors)) + 
  theme_bw()
wUNI_planted_pcoa

32.1% variation explained by the Weighted Unifrac PCoA

Combine PCoAs

#Let’s plot all three together into one plot to have a concise visualization of the three metrics.

(soren_n_pcoa + theme(legend.position = "none")) + 
  (bray_n_pcoa + theme(legend.position = "none")) + 
    (wUNI_n_pcoa + theme(legend.position = "none"))

(soren_planted_pcoa + theme(legend.position = "none")) + 
  (bray_planted_pcoa + theme(legend.position = "none")) + 
    (wUNI_planted_pcoa + theme(legend.position = "none"))

NMDS

Weighted Unifrac

Since we did 3 of the dissimilarity metrics for the PCoA, let’s just plot one example of them for the NMDS plotting. Here, we will use weighted Unifrac

# Calculate the Weighted Unifrac distance
scaled_wUNI_nmds <- 
  ordinate(
    physeq = scaled_rooted_physeq,
    method = "NMDS",
    distance = "wunifrac")

## Run 0 stress 0.1777991 
## Run 1 stress 0.1828857 
## Run 2 stress 0.1762887 
## ... New best solution
## ... Procrustes: rmse 0.1202383  max resid 0.2898712 
## Run 3 stress 0.1846833 
## Run 4 stress 0.1730309 
## ... New best solution
## ... Procrustes: rmse 0.1020346  max resid 0.2983786 
## Run 5 stress 0.176289 
## Run 6 stress 0.1822507 
## Run 7 stress 0.1809346 
## Run 8 stress 0.1740514 
## Run 9 stress 0.1887748 
## Run 10 stress 0.1802961 
## Run 11 stress 0.1841897 
## Run 12 stress 0.1732299 
## ... Procrustes: rmse 0.1217492  max resid 0.5223252 
## Run 13 stress 0.1729327 
## ... New best solution
## ... Procrustes: rmse 0.03561431  max resid 0.1410735 
## Run 14 stress 0.1790464 
## Run 15 stress 0.1876594 
## Run 16 stress 0.1890485 
## Run 17 stress 0.1719995 
## ... New best solution
## ... Procrustes: rmse 0.05067667  max resid 0.2404631 
## Run 18 stress 0.182121 
## Run 19 stress 0.178459 
## Run 20 stress 0.1775848 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     18: stress ratio > sratmax
##      2: scale factor of the gradient < sfgrmin

# Plot the PCoA
wUNI_n_nmds <- 
  plot_ordination(
    physeq = scaled_rooted_physeq,
    ordination = scaled_wUNI_nmds,
    color = as.factor("n_level"),
    title = "Weighted Unifrac NMDS") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(n_level))) +
  scale_color_manual(values = as.factor(n_colors)) + 
  theme_bw()
wUNI_n_nmds

wUNI_planted_nmds <- 
  plot_ordination(
    physeq = scaled_rooted_physeq,
    ordination = scaled_wUNI_nmds,
    color = as.factor("planted"),
    title = "Weighted Unifrac NMDS") +
  geom_point(size=5, alpha = 0.5, aes(color = as.factor(planted))) +
  scale_color_manual(values = as.factor(planted_colors)) + 
  theme_bw()
wUNI_planted_nmds

(wUNI_n_pcoa + theme(legend.position = "none")) + 
  (wUNI_n_nmds + theme(legend.position = "none"))

(wUNI_planted_pcoa + theme(legend.position = "none")) + 
  (wUNI_planted_nmds + theme(legend.position = "none"))

Statistical Significance Testing

PERMANOVA

# Calculate all three of the distance matrices
scaled_sorensen_dist <- phyloseq::distance(scaled_rooted_physeq, method = "bray", binary = TRUE)
scaled_bray_dist <- phyloseq::distance(scaled_rooted_physeq, method = "bray")
scaled_wUnifrac_dist <- phyloseq::distance(scaled_rooted_physeq, method = "wunifrac")

# make a data frame from the sample_data
# All distance matrices will be the same metadata because they 
# originate from the same phyloseq object. 
metadata <- data.frame(sample_data(scaled_rooted_physeq))

# Adonis test
# In this example we are testing the hypothesis that the five stations
# that were collected have different centroids in the ordination space 
# for each of the dissimilarity metrics, we are using a discrete variable 
adonis2(scaled_sorensen_dist ~ n_level, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_sorensen_dist ~ n_level, data = metadata)
##          Df SumOfSqs      R2      F Pr(>F)  
## n_level   1   0.2398 0.03253 1.1095  0.031 *
## Residual 33   7.1334 0.96747                
## Total    34   7.3732 1.00000                
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

adonis2(scaled_bray_dist ~ n_level, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_bray_dist ~ n_level, data = metadata)
##          Df SumOfSqs    R2      F Pr(>F)
## n_level   1   0.1126 0.033 1.1261  0.191
## Residual 33   3.2989 0.967              
## Total    34   3.4115 1.000

adonis2(scaled_wUnifrac_dist ~ n_level, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_wUnifrac_dist ~ n_level, data = metadata)
##          Df SumOfSqs      R2      F Pr(>F)
## n_level   1  0.01372 0.02958 1.0059  0.372
## Residual 33  0.45014 0.97042              
## Total    34  0.46386 1.00000

adonis2(scaled_sorensen_dist ~ planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_sorensen_dist ~ planted, data = metadata)
##          Df SumOfSqs      R2      F Pr(>F)    
## planted   1   0.3654 0.04955 1.7205  0.001 ***
## Residual 33   7.0078 0.95045                  
## Total    34   7.3732 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

adonis2(scaled_bray_dist ~ planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_bray_dist ~ planted, data = metadata)
##          Df SumOfSqs      R2      F Pr(>F)    
## planted   1   0.3631 0.10644 3.9308  0.001 ***
## Residual 33   3.0484 0.89356                  
## Total    34   3.4115 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

adonis2(scaled_wUnifrac_dist ~ planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_wUnifrac_dist ~ planted, data = metadata)
##          Df SumOfSqs      R2      F Pr(>F)    
## planted   1  0.06930 0.14939 5.7958  0.001 ***
## Residual 33  0.39456 0.85061                  
## Total    34  0.46386 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

No significant diffrence between nitrogen levels

Differences between planted and unplanted treatments are significant. The most difference is explained by the Weighted Unifrac.

# We might also care about other variables
# Here, we will add date and fraction as variables
# multiplicative model ORDER MATTERS! 
adonis2(scaled_sorensen_dist ~ n_level * growth_stage * planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_sorensen_dist ~ n_level * growth_stage * planted, data = metadata)
##                              Df SumOfSqs      R2      F Pr(>F)    
## n_level                       1   0.2398 0.03253 1.1499  0.011 *  
## growth_stage                  1   0.2316 0.03142 1.1106  0.039 *  
## planted                       1   0.3648 0.04948 1.7492  0.001 ***
## n_level:growth_stage          1   0.2242 0.03041 1.0751  0.088 .  
## n_level:planted               1   0.2233 0.03029 1.0708  0.104    
## growth_stage:planted          1   0.2378 0.03226 1.1403  0.022 *  
## n_level:growth_stage:planted  1   0.2202 0.02987 1.0560  0.151    
## Residual                     27   5.6313 0.76375                  
## Total                        34   7.3732 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

adonis2(scaled_bray_dist ~ n_level * growth_stage * planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_bray_dist ~ n_level * growth_stage * planted, data = metadata)
##                              Df SumOfSqs      R2      F Pr(>F)    
## n_level                       1   0.1126 0.03300 1.2926  0.055 .  
## growth_stage                  1   0.1308 0.03835 1.5023  0.018 *  
## planted                       1   0.3651 0.10701 4.1915  0.001 ***
## n_level:growth_stage          1   0.1008 0.02954 1.1572  0.160    
## n_level:planted               1   0.1138 0.03336 1.3066  0.053 .  
## growth_stage:planted          1   0.1346 0.03945 1.5452  0.012 *  
## n_level:growth_stage:planted  1   0.1023 0.02999 1.1749  0.124    
## Residual                     27   2.3516 0.68930                  
## Total                        34   3.4115 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

# Note that the ORDER MATTERS!
adonis2(scaled_wUnifrac_dist ~ n_level * growth_stage * planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_wUnifrac_dist ~ n_level * growth_stage * planted, data = metadata)
##                              Df SumOfSqs      R2      F Pr(>F)    
## n_level                       1  0.01372 0.02958 1.2941  0.171    
## growth_stage                  1  0.03026 0.06524 2.8539  0.003 ** 
## planted                       1  0.07006 0.15103 6.6069  0.001 ***
## n_level:growth_stage          1  0.01150 0.02480 1.0849  0.295    
## n_level:planted               1  0.01511 0.03257 1.4250  0.108    
## growth_stage:planted          1  0.02371 0.05111 2.2361  0.011 *  
## n_level:growth_stage:planted  1  0.01321 0.02848 1.2458  0.165    
## Residual                     27  0.28629 0.61719                  
## Total                        34  0.46386 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

adonis2(scaled_wUnifrac_dist ~ growth_stage * n_level * planted, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## adonis2(formula = scaled_wUnifrac_dist ~ growth_stage * n_level * planted, data = metadata)
##                              Df SumOfSqs      R2      F Pr(>F)    
## growth_stage                  1  0.03057 0.06591 2.8834  0.002 ** 
## n_level                       1  0.01341 0.02891 1.2646  0.171    
## planted                       1  0.07006 0.15103 6.6069  0.001 ***
## growth_stage:n_level          1  0.01150 0.02480 1.0849  0.321    
## growth_stage:planted          1  0.02447 0.05276 2.3080  0.007 ** 
## n_level:planted               1  0.01435 0.03093 1.3531  0.133    
## growth_stage:n_level:planted  1  0.01321 0.02848 1.2458  0.197    
## Residual                     27  0.28629 0.61719                  
## Total                        34  0.46386 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

BetaDispR

The PERMANOVA is sensitive to variance/dispersion in the data. Therefore, we need to run a homogeneity of dispersion test to test for the sensitivity of our PERMANOVA results to variance.

# Homogeneity of Dispersion test with beta dispr
# Sorensen 
beta_soren_n <- betadisper(scaled_sorensen_dist, metadata$n_level)
permutest(beta_soren_n)

## 
## Permutation test for homogeneity of multivariate dispersions
## Permutation: free
## Number of permutations: 999
## 
## Response: Distances
##           Df    Sum Sq   Mean Sq      F N.Perm Pr(>F)   
## Groups     2 0.0068845 0.0034422 6.1052    999  0.002 **
## Residuals 32 0.0180421 0.0005638                        
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

beta_soren_planted <- betadisper(scaled_sorensen_dist, metadata$planted)
permutest(beta_soren_planted)

## 
## Permutation test for homogeneity of multivariate dispersions
## Permutation: free
## Number of permutations: 999
## 
## Response: Distances
##           Df    Sum Sq    Mean Sq      F N.Perm Pr(>F)
## Groups     1 0.0000275 0.00002755 0.0309    999  0.868
## Residuals 33 0.0293858 0.00089048

# Bray-curtis 
beta_bray_n <- betadisper(scaled_bray_dist, metadata$n_level)
permutest(beta_bray_n)

## 
## Permutation test for homogeneity of multivariate dispersions
## Permutation: free
## Number of permutations: 999
## 
## Response: Distances
##           Df   Sum Sq   Mean Sq      F N.Perm Pr(>F)  
## Groups     2 0.006049 0.0030245 2.6353    999   0.08 .
## Residuals 32 0.036727 0.0011477                       
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

beta_bray_planted <- betadisper(scaled_bray_dist, metadata$planted)
permutest(beta_bray_planted)

## 
## Permutation test for homogeneity of multivariate dispersions
## Permutation: free
## Number of permutations: 999
## 
## Response: Distances
##           Df   Sum Sq    Mean Sq      F N.Perm Pr(>F)
## Groups     1 0.000271 0.00027128 0.1672    999  0.703
## Residuals 33 0.053533 0.00162221

# Weighted Unifrac 
beta_bray_n <- betadisper(scaled_wUnifrac_dist, metadata$n_level)
permutest(beta_bray_n)

## 
## Permutation test for homogeneity of multivariate dispersions
## Permutation: free
## Number of permutations: 999
## 
## Response: Distances
##           Df    Sum Sq    Mean Sq      F N.Perm Pr(>F)
## Groups     2 0.0008306 0.00041531 0.9309    999  0.395
## Residuals 32 0.0142763 0.00044613

beta_bray_planted <- betadisper(scaled_wUnifrac_dist, metadata$planted)
permutest(beta_bray_planted)

## 
## Permutation test for homogeneity of multivariate dispersions
## Permutation: free
## Number of permutations: 999
## 
## Response: Distances
##           Df    Sum Sq    Mean Sq      F N.Perm Pr(>F)
## Groups     1 0.0000012 0.00000121 0.0022    999  0.963
## Residuals 33 0.0178210 0.00054003

Variance of Sorensen influenced by nitrogen level.

Taxonomic Composition

Phylum

# Set the phylum colors
phylum_colors <- c(
  Acidobacteriota = "navy", 
  Actinobacteria = "purple",
  Gemmatimonadota = "orange",
  Patescibacteria = "lightpink",
  Firmacutes = "darkslategray2", 
  Armatimonadota = "deeppink1",
  Alphaproteobacteria = "plum2", 
  Bacteroidota = "gold", 
  Betaproteobacteria = "plum1", 
  Bdellovibrionota = "red1",
  Chloroflexi="black", 
  Crenarchaeota = "firebrick",
  Cyanobacteria = "limegreen",
  Deltaproteobacteria = "grey", 
  Desulfobacterota="magenta",
  Firmicutes = "#3E9B96",
  Gammaproteobacteria = "greenyellow",
  "Marinimicrobia (SAR406 clade)" = "yellow",
  Myxococcota = "#B5D6AA",
  Nitrospirota = "palevioletred1",
  Proteobacteria = "royalblue",
  Planctomycetota = "darkorange", 
  "SAR324 clade(Marine group B)" = "olivedrab",
  "WPS-2"= "greenyellow",
  Thermoplasmatota = "green",
  Verrucomicrobiota = "darkorchid1")
 # Other = "grey")

# Calculate the phylum relative abundance 
# Note: The read depth MUST be normalized in some way: scale_reads
phylum_df <- 
  scaled_rooted_physeq %>%
  # agglomerate at the phylum level 
  tax_glom(taxrank = "Phylum") %>% 
  # Transform counts to relative abundance 
  transform_sample_counts(function (x) {x/sum(x)}) %>%
  # Melt to a long format 
  psmelt() %>%
  # Filter out Phyla that are <1% - get rid of low abundant Phyla
  dplyr::filter(Abundance > 0.01)

phylum_df %>%
  # Warning: It's important to have one sample per x value, 
  # otherwise, it will take the sum between multiple samples
  ggplot(aes(x = planted, y = Abundance, fill = Phylum)) + 
  facet_grid(Phylum~growth_stage~n_level, scale = "free") + 
  geom_bar(stat = "identity", color = "black") + 
  labs(title = "Surface Phylum Composition") + 
  scale_fill_manual(values = phylum_colors) + 
  theme_bw() + 
  theme(axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1),
        strip.text.y = element_text(size = 5, angle = 90))

# Narrow in on a specific group
# Firmacutes - y: abundance, x: station, dot plot + boxplot
phylum_df %>%
  dplyr::filter(Phylum == "Firmicutes") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = planted, color = planted)) + 
  facet_wrap(.~Phylum, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Firmicutes Phylum Abundance", ylab = "Planted") + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1),
          legend.position = "right")

# Statistically: Kruskall-Wallis followed by a Tukey's Posthoc test
# These are non-parametric (non-normal) stat tests

Family

# Calculate the Family relative abundance 
# Note: The read depth MUST be normalized in some way: scale_reads
family_df <- 
  scaled_rooted_physeq %>%
  # agglomerate at the phylum level 
  tax_glom(taxrank = "Family") %>% 
  # Transform counts to relative abundance 
  transform_sample_counts(function (x) {x/sum(x)}) %>%
  # Melt to a long format 
  psmelt() %>%
  # Filter out Phyla that are <1% - get rid of low abundant Phyla
  dplyr::filter(Abundance > 0.01)
  
  # Check family_df
#str(family_df)

family_df %>%
  dplyr::filter(Phylum == "Firmicutes") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = planted, color = planted)) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Firmicutes Family Abundance", ylab = "Planted") + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1),
          legend.position = "right")

#Caulobacteraceae - keystone taxa identified by Cui et al.for sampling on day 30.
family_df %>%
  dplyr::filter(Family == "Caulobacteraceae") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = planted, color = planted)) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Caulobacteraceae Family Abundance Planted vs. Unplanted", ylab = "Planted") + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1),
          legend.position = "right")

family_df %>%
  dplyr::filter(Family == "Caulobacteraceae") %>%
  # build the plot 
  ggplot(aes(x = growth_stage, y = Abundance, 
             fill = as.factor(growth_stage), color = as.factor(growth_stage))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Caulobacteraceae Family Abundance", xlab = ("Growth Stage")) + 
  scale_color_manual(values = growth_stage_colors) + 
  scale_fill_manual(values = growth_stage_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "right", legend.title=element_blank())

#Chitinophagaceae - keystone taxa identified by Cui et al.for sampling on day 30.
family_df %>%
  dplyr::filter(Family == "Chitinophagaceae") %>%
  # build the plot 
  ggplot(aes(x = growth_stage, y = Abundance, 
             fill = as.factor(growth_stage), color = as.factor(growth_stage))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Chitinophagaceae Family Abundance", xlab = ("Growth Stage")) + 
  scale_color_manual(values = growth_stage_colors) + 
  scale_fill_manual(values = growth_stage_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "right", legend.title=element_blank())

family_df %>%
  dplyr::filter(Family == "Chitinophagaceae") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = planted, color = planted)) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Chitinophagaceae Family Abundance Planted vs. Unplanted", xlab = ("Planted")) + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "right", legend.title=element_blank())

# Xanthobacteraceae - keystone taxa identified by Cui et al.for sampling on day 75
family_df %>%
  dplyr::filter(Family == "Xanthobacteraceae") %>%
  # build the plot 
  ggplot(aes(x = growth_stage, y = Abundance, 
             fill = as.factor(growth_stage), color = as.factor(growth_stage))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Xanthobacteraceae Family Abundance", xlab = ("Growth Stage")) + 
  scale_color_manual(values = growth_stage_colors) + 
  scale_fill_manual(values = growth_stage_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "right", legend.title=element_blank())

family_df %>%
  dplyr::filter(Family == "Xanthobacteraceae") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = planted, color = planted)) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Xanthobacteraceae Family Abundance Planted vs. Unplanted", xlab = ("Planted")) + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "right", legend.title=element_blank())

Genus

# Calculate the Family relative abundance 
# Note: The read depth MUST be normalized in some way: scale_reads
genus_df <- 
  scaled_rooted_physeq %>%
  # agglomerate at the phylum level 
  tax_glom(taxrank = "Genus") %>% 
  # Transform counts to relative abundance 
  transform_sample_counts(function (x) {x/sum(x)}) %>%
  # Melt to a long format 
  psmelt() %>%
  # Filter out Phyla that are <1% - get rid of low abundant Phyla
  dplyr::filter(Abundance > 0.01)

genus_df %>%
  dplyr::filter(Phylum == "Firmicutes") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = planted, color = planted)) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Firmicutes Genus Abundance", ylab = "Planted") + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(angle = 30, hjust = 1, vjust = 1),
          legend.position = "right")

#Bacillus - keystone taxa identified by Cui et al.for sampling on day 30
genus_df %>%
  dplyr::filter(Genus == "Bacillus") %>%
  # build the plot 
  ggplot(aes(x = growth_stage, y = Abundance, 
             fill = as.factor(growth_stage), color = as.factor(growth_stage))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Bacillus Genus Abundance", xlab = ("Growth Stage")) + 
  scale_color_manual(values = growth_stage_colors) + 
  scale_fill_manual(values = growth_stage_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "bottom", legend.title=element_blank())

genus_df %>%
  dplyr::filter(Genus == "Bacillus") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = as.factor(planted), color = as.factor(planted))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Bacillus Genus Abundance Planted vs. Unplanted", xlab = ("Planted")) + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "bottom", legend.title=element_blank())

#Gemmatimonas - keystone taxa identified by Cui et al.for sampling on day 75

genus_df %>%
  dplyr::filter(Genus == "Gemmatimonas") %>%
  # build the plot 
  ggplot(aes(x = growth_stage, y = Abundance, 
             fill = as.factor(growth_stage), color = as.factor(growth_stage))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Gemmatimonas Genus Abundance", xlab = ("Growth Stage")) + 
  scale_color_manual(values = growth_stage_colors) + 
  scale_fill_manual(values = growth_stage_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "bottom", legend.title=element_blank())

genus_df %>%
  dplyr::filter(Genus == "Gemmatimonas") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = as.factor(planted), color = as.factor(planted))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Gemmatimonas Genus Abundance Planted vs", xlab = ("Planted")) + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "bottom", legend.title=element_blank())

#Candidatus Koribacter - keystone taxa identified by Cui et al.for sampling on day 75

genus_df %>%
  dplyr::filter(Genus == "Candidatus Koribacter") %>%
  # build the plot 
  ggplot(aes(x = growth_stage, y = Abundance, 
             fill = as.factor(growth_stage), color = as.factor(growth_stage))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Candidatus Koribacter Genus Abundance", xlab = ("Growth Stage")) + 
  scale_color_manual(values = growth_stage_colors) + 
  scale_fill_manual(values = growth_stage_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "bottom", legend.title=element_blank())

genus_df %>%
  dplyr::filter(Genus == "Candidatus Koribacter") %>%
  # build the plot 
  ggplot(aes(x = planted, y = Abundance, 
             fill = as.factor(planted), color = as.factor(planted))) + 
  facet_wrap(.~Family, scales = "free_y", nrow = 1) + 
  geom_boxplot(alpha = 0.5, outlier.shape = NA) + # outliers not plotted here in boxplot 
  geom_jitter() + 
  theme_bw() + 
  labs(title = "Candidatus Koribacter Genus Abundance Planted vs", xlab = ("Planted")) + 
  scale_color_manual(values = planted_colors) + 
  scale_fill_manual(values = planted_colors) + 
    theme(axis.text.x = element_text(),
          legend.position = "bottom", legend.title=element_blank())

Session Information

For reproducibility

devtools::session_info()

## ─ Session info ───────────────────────────────────────────────────────────────
##  setting  value
##  version  R version 4.3.2 (2023-10-31)
##  os       Rocky Linux 9.0 (Blue Onyx)
##  system   x86_64, linux-gnu
##  ui       X11
##  language (EN)
##  collate  en_US.UTF-8
##  ctype    en_US.UTF-8
##  tz       America/New_York
##  date     2024-04-30
##  pandoc   3.1.1 @ /usr/lib/rstudio-server/bin/quarto/bin/tools/ (via rmarkdown)
## 
## ─ Packages ───────────────────────────────────────────────────────────────────
##  package          * version   date (UTC) lib source
##  ade4               1.7-22    2023-02-06 [1] CRAN (R 4.3.2)
##  ape                5.7-1     2023-03-13 [2] CRAN (R 4.3.2)
##  Biobase            2.62.0    2023-10-24 [2] Bioconductor
##  BiocGenerics       0.48.1    2023-11-01 [2] Bioconductor
##  biomformat         1.30.0    2023-10-24 [1] Bioconductor
##  Biostrings         2.70.1    2023-10-25 [2] Bioconductor
##  bitops             1.0-7     2021-04-24 [2] CRAN (R 4.3.2)
##  bslib              0.5.1     2023-08-11 [2] CRAN (R 4.3.2)
##  cachem             1.0.8     2023-05-01 [2] CRAN (R 4.3.2)
##  callr              3.7.3     2022-11-02 [2] CRAN (R 4.3.2)
##  cli                3.6.1     2023-03-23 [2] CRAN (R 4.3.2)
##  cluster            2.1.4     2022-08-22 [2] CRAN (R 4.3.2)
##  codetools          0.2-19    2023-02-01 [2] CRAN (R 4.3.2)
##  colorspace         2.1-0     2023-01-23 [2] CRAN (R 4.3.2)
##  crayon             1.5.2     2022-09-29 [2] CRAN (R 4.3.2)
##  data.table         1.14.8    2023-02-17 [2] CRAN (R 4.3.2)
##  devtools         * 2.4.5     2022-10-11 [1] CRAN (R 4.3.2)
##  digest             0.6.33    2023-07-07 [2] CRAN (R 4.3.2)
##  dplyr            * 1.1.3     2023-09-03 [2] CRAN (R 4.3.2)
##  ellipsis           0.3.2     2021-04-29 [2] CRAN (R 4.3.2)
##  evaluate           0.23      2023-11-01 [2] CRAN (R 4.3.2)
##  fansi              1.0.5     2023-10-08 [2] CRAN (R 4.3.2)
##  farver             2.1.1     2022-07-06 [2] CRAN (R 4.3.2)
##  fastmap            1.1.1     2023-02-24 [2] CRAN (R 4.3.2)
##  forcats          * 1.0.0     2023-01-29 [1] CRAN (R 4.3.2)
##  foreach            1.5.2     2022-02-02 [2] CRAN (R 4.3.2)
##  fs                 1.6.3     2023-07-20 [2] CRAN (R 4.3.2)
##  generics           0.1.3     2022-07-05 [2] CRAN (R 4.3.2)
##  GenomeInfoDb       1.38.0    2023-10-24 [2] Bioconductor
##  GenomeInfoDbData   1.2.11    2023-11-07 [2] Bioconductor
##  ggplot2          * 3.5.0     2024-02-23 [2] CRAN (R 4.3.2)
##  glue               1.6.2     2022-02-24 [2] CRAN (R 4.3.2)
##  gtable             0.3.4     2023-08-21 [2] CRAN (R 4.3.2)
##  highr              0.10      2022-12-22 [2] CRAN (R 4.3.2)
##  hms                1.1.3     2023-03-21 [1] CRAN (R 4.3.2)
##  htmltools          0.5.7     2023-11-03 [2] CRAN (R 4.3.2)
##  htmlwidgets        1.6.2     2023-03-17 [2] CRAN (R 4.3.2)
##  httpuv             1.6.12    2023-10-23 [2] CRAN (R 4.3.2)
##  igraph             1.5.1     2023-08-10 [2] CRAN (R 4.3.2)
##  IRanges            2.36.0    2023-10-24 [2] Bioconductor
##  iterators          1.0.14    2022-02-05 [2] CRAN (R 4.3.2)
##  jquerylib          0.1.4     2021-04-26 [2] CRAN (R 4.3.2)
##  jsonlite           1.8.7     2023-06-29 [2] CRAN (R 4.3.2)
##  knitr              1.45      2023-10-30 [2] CRAN (R 4.3.2)
##  labeling           0.4.3     2023-08-29 [2] CRAN (R 4.3.2)
##  later              1.3.1     2023-05-02 [2] CRAN (R 4.3.2)
##  lattice          * 0.21-9    2023-10-01 [2] CRAN (R 4.3.2)
##  lifecycle          1.0.3     2022-10-07 [2] CRAN (R 4.3.2)
##  lubridate        * 1.9.3     2023-09-27 [1] CRAN (R 4.3.2)
##  magrittr           2.0.3     2022-03-30 [2] CRAN (R 4.3.2)
##  MASS               7.3-60    2023-05-04 [2] CRAN (R 4.3.2)
##  Matrix             1.6-1.1   2023-09-18 [2] CRAN (R 4.3.2)
##  memoise            2.0.1     2021-11-26 [2] CRAN (R 4.3.2)
##  mgcv               1.9-0     2023-07-11 [2] CRAN (R 4.3.2)
##  mime               0.12      2021-09-28 [2] CRAN (R 4.3.2)
##  miniUI             0.1.1.1   2018-05-18 [2] CRAN (R 4.3.2)
##  multtest           2.58.0    2023-10-24 [1] Bioconductor
##  munsell            0.5.0     2018-06-12 [2] CRAN (R 4.3.2)
##  nlme               3.1-163   2023-08-09 [2] CRAN (R 4.3.2)
##  pacman             0.5.1     2019-03-11 [1] CRAN (R 4.3.2)
##  patchwork        * 1.2.0     2024-01-08 [1] CRAN (R 4.3.2)
##  permute          * 0.9-7     2022-01-27 [1] CRAN (R 4.3.2)
##  phyloseq         * 1.41.1    2024-03-13 [1] Github (joey711/phyloseq@c260561)
##  pillar             1.9.0     2023-03-22 [2] CRAN (R 4.3.2)
##  pkgbuild           1.4.2     2023-06-26 [2] CRAN (R 4.3.2)
##  pkgconfig          2.0.3     2019-09-22 [2] CRAN (R 4.3.2)
##  pkgload            1.3.3     2023-09-22 [2] CRAN (R 4.3.2)
##  plyr               1.8.9     2023-10-02 [2] CRAN (R 4.3.2)
##  prettyunits        1.2.0     2023-09-24 [2] CRAN (R 4.3.2)
##  processx           3.8.2     2023-06-30 [2] CRAN (R 4.3.2)
##  profvis            0.3.8     2023-05-02 [2] CRAN (R 4.3.2)
##  promises           1.2.1     2023-08-10 [2] CRAN (R 4.3.2)
##  ps                 1.7.5     2023-04-18 [2] CRAN (R 4.3.2)
##  purrr            * 1.0.2     2023-08-10 [2] CRAN (R 4.3.2)
##  R6                 2.5.1     2021-08-19 [2] CRAN (R 4.3.2)
##  Rcpp               1.0.11    2023-07-06 [2] CRAN (R 4.3.2)
##  RCurl              1.98-1.13 2023-11-02 [2] CRAN (R 4.3.2)
##  readr            * 2.1.5     2024-01-10 [1] CRAN (R 4.3.2)
##  remotes            2.4.2.1   2023-07-18 [2] CRAN (R 4.3.2)
##  reshape2           1.4.4     2020-04-09 [2] CRAN (R 4.3.2)
##  rhdf5              2.46.1    2023-11-29 [1] Bioconductor 3.18 (R 4.3.2)
##  rhdf5filters       1.14.1    2023-11-06 [1] Bioconductor
##  Rhdf5lib           1.24.2    2024-02-07 [1] Bioconductor 3.18 (R 4.3.2)
##  rlang              1.1.2     2023-11-04 [2] CRAN (R 4.3.2)
##  rmarkdown          2.25      2023-09-18 [2] CRAN (R 4.3.2)
##  rstudioapi         0.15.0    2023-07-07 [2] CRAN (R 4.3.2)
##  S4Vectors          0.40.1    2023-10-26 [2] Bioconductor
##  sass               0.4.7     2023-07-15 [2] CRAN (R 4.3.2)
##  scales             1.3.0     2023-11-28 [2] CRAN (R 4.3.2)
##  sessioninfo        1.2.2     2021-12-06 [2] CRAN (R 4.3.2)
##  shiny              1.7.5.1   2023-10-14 [2] CRAN (R 4.3.2)
##  stringi            1.7.12    2023-01-11 [2] CRAN (R 4.3.2)
##  stringr          * 1.5.0     2022-12-02 [2] CRAN (R 4.3.2)
##  survival           3.5-7     2023-08-14 [2] CRAN (R 4.3.2)
##  tibble           * 3.2.1     2023-03-20 [2] CRAN (R 4.3.2)
##  tidyr            * 1.3.0     2023-01-24 [2] CRAN (R 4.3.2)
##  tidyselect         1.2.0     2022-10-10 [2] CRAN (R 4.3.2)
##  tidyverse        * 2.0.0     2023-02-22 [1] CRAN (R 4.3.2)
##  timechange         0.3.0     2024-01-18 [1] CRAN (R 4.3.2)
##  tzdb               0.4.0     2023-05-12 [1] CRAN (R 4.3.2)
##  urlchecker         1.0.1     2021-11-30 [2] CRAN (R 4.3.2)
##  usethis          * 2.2.2     2023-07-06 [2] CRAN (R 4.3.2)
##  utf8               1.2.4     2023-10-22 [2] CRAN (R 4.3.2)
##  vctrs              0.6.4     2023-10-12 [2] CRAN (R 4.3.2)
##  vegan            * 2.6-4     2022-10-11 [1] CRAN (R 4.3.2)
##  withr              2.5.2     2023-10-30 [2] CRAN (R 4.3.2)
##  xfun               0.41      2023-11-01 [2] CRAN (R 4.3.2)
##  xtable             1.8-4     2019-04-21 [2] CRAN (R 4.3.2)
##  XVector            0.42.0    2023-10-24 [2] Bioconductor
##  yaml               2.3.7     2023-01-23 [2] CRAN (R 4.3.2)
##  zlibbioc           1.48.0    2023-10-24 [2] Bioconductor
## 
##  [1] /home/alm379/R/x86_64-pc-linux-gnu-library/4.3
##  [2] /programs/R-4.3.2/library
## 
## ──────────────────────────────────────────────────────────────────────────────

05_CommunityAnalysis.Rmd

Andrea Martinez

2024-04-30

Goals

Setup

Set the seed

Load Libraries

Load Data

Explore Read Counts

Raw read depth

Remove lowly seq sample

Normalize read counts

Scale the reads and check the distribution of the seq depth

Cacluate & Visualize community dissimilarity

PCoA

Sorensen

Bray

Weighted Unifrac

Combine PCoAs

NMDS

Weighted Unifrac

Statistical Significance Testing

PERMANOVA

BetaDispR

Taxonomic Composition

Phylum

Family

Genus

Session Information